Pluris tem dispase 2

Cortical differentiation from human ESC was performed using previously described protocol (Espuny-camacho et al., 2013) with slight modifications. At differentiation day -8, the hES cells were dissociated using PluriSTEM Dispase-II (Merck)/ Collagenase (Thermo Fisher Scientific) solution and plated on Growth factor-reduced matrigel (Corning) in Essential 8 Flex medium (Thermo Fisher Scientific). At differentiation day -2, the cells were dissociated using Stem-Pro Accutase (Thermo Fisher Scientific) and plated at low confluency (5,000-10,000 cells/cm 2 ) on hES qualified matrigel (Corning)
using Essential 8 Flex medium supplemented with 10 µM ROCK inhibitor (Y-27632; Merck). At differentiation day 0, the medium was changed to DDM (Gaspard et al., 2008) supplemented with B27 (Thermo Fisher Scientific) and 100 ng/mL Noggin (R&D systems), and the medium was replenished every day. After 16 days of differentiation, the medium was changed to DDM, supplemented with B27 (DDM/B27), and changed every day. At day 25, the progenitors were dissociated using Accutase and frozen using mFreSR (Stemcell technologies). Cortical progenitors were thawed on matrigelcoated coverslips 12-well using DDM/B27 supplemented with 10 µM ROCK inhibitor and fixed after 6 days using 4% paraformaldehyde for 20 min at 4° or ice-cold methanol for 6 min and then rinsed 3 times with PBS before immunostaining.